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Cell-free biosensors can detect various molecules, thus promising to transform the landscape of diagnostics. Here, we developed a simple, rapid, sensitive, and field-deployable small-molecule detection platform based on allosteric transcription factor (aTF)-regulated expression of a clustered regularly interspaced short palindromic repeats (CRISPR) array coupled to Cas12a activity. To this end, we engineered an expression cassette harboring a T7 promoter, an aTF binding sequence, a Cas12a CRISPR array, and protospacer adjacent motif-flanked Cas12a target sequences. In the presence of the ligand, dissociation of the aTF allows transcription of the CRISPR array; this leads to activation of Cas12a collateral activity, which cleaves a single-stranded DNA linker to free a quenched fluorophore, resulting in a rapid, significant increase of fluorescence. As a proof of concept, we used TetR as the aTF to detect different tetracycline antibiotics with high sensitivity and specificity and a simple, hand-held visualizer to develop a fluorescence-based visual readout. We also adapted a mobile phone application to further simplify the interpretation of the results. Finally, we showed that the reagents could be lyophilized to facilitate storage and distribution. This detection platform represents a valuable addition to the toolbox of cell-free, CRISPR-based biosensors, with great potential for in-field deployment to detect non-nucleic acid small molecules.

Pigmented rice (Oryza sativa L.) is a rich source of nutrients, but pigmented lines typically have long life cycles and limited productivity. Here we generated genome assemblies of 5 pigmented rice varieties and evaluated the genetic variation among 51 pigmented rice varieties by resequencing an additional 46 varieties. Phylogenetic analyses divided the pigmented varieties into four varietal groups: Geng-japonica, Xian-indica, circum-Aus and circum-Basmati. Metabolomics and ionomics profiling revealed that black rice varieties are rich in aromatic secondary metabolites. We established a regeneration and transformation system and used CRISPR–Cas9 to knock out three flowering time repressors (Hd2, Hd4 and Hd5) in the black Indonesian rice Cempo Ireng, resulting in an early maturing variety with shorter stature. Our study thus provides a multi-omics resource for understanding and improving Asian pigmented rice.

Zero hunger is one of the sustainable development goals set by the United Nations in 2015 to achieve global food security by 2030. The current harvest of crops is insufficient; feeding the world's population and meeting the goal of zero hunger by 2030 will require larger and more consistent crop production. Clustered regularly interspaced short palindromic repeats-associated protein (CRISPR-Cas) technology is widely used for the plant genome editing. In this review, we consider this technology as a potential tool for achieving zero hunger. We provide a comprehensive overview of CRISPR-Cas technology and its most important applications for food crops' improvement. We also conferred current and potential technological breakthroughs that will help in breeding future crops to end global hunger. The regulatory aspects of deploying this technology in commercial sectors, bioethics, and the production of transgene-free plants are also discussed. We hope that the CRISPR-Cas system will accelerate the breeding of improved crop cultivars compared with conventional breeding and pave the way toward the zero hunger goal.

Rapid, sensitive, and specific point-of-care testing for pathogens is crucial for disease control. Lateral flow assays (LFAs) have been employed for nucleic acid detection, but they have limited sensitivity and specificity. Here, we used a fusion of catalytically inactive SpCas9 endonuclease and VirD2 relaxase for sensitive, specific nucleic acid detection by LFA. In this assay, the target nucleic acid is amplified with biotinylated oligos. VirD2-dCas9 specifically binds the target sequence via dCas9 and covalently binds to a FAM-tagged oligonucleotide via VirD2. The biotin label and FAM tag are detected by a commercially available LFA. We coupled this system, named Vigilant (VirD2-dCas9 guided and LFA-coupled nucleic acid test), to reverse transcription-recombinase polymerase amplification to detect SARS-CoV2 in clinical samples. Vigilant exhibited a limit of detection of 2.5 copies/μL, comparable to CRISPR-based systems, and showed no cross-reactivity with SARS-CoV1 or MERS. Vigilant offers an easy-to-use, rapid, cost-effective, and robust detection platform for SARS-CoV2.

Synthetic directed evolution via localized sequence diversification and the simultaneous application of selection pressure is a promising method for producing new, beneficial alleles that affect traits of interest in diverse species; however, this technique has rarely been applied in plants. Developing systems to induce localized sequence diversification at high efficiency will expand our ability to evolve traits of interest that improve global food security. In this study, we designed, built, and tested a chimeric fusion of T7 RNA Polymerase (RNAP) and deaminase to enable the localized sequence diversification of a target sequence of interest. We tested our T7 RNAP–DNA base editor in Nicotiana benthamiana transient assays to target a transgene expressing GFP under the control of the T7 promoter. More than 7% of C nucleotides were converted to T in long segments of the GFP sequence. We then targeted the T7 promoter-driven ACETOLACTATE SYNTHASE (ALS) sequence that had been stably integrated in the rice (Oryza sativa) genome and generated C-to-T and G-to-A transitions. We used herbicide treatment as selection pressure for the evolution of the ALS sequence, resulting in the enrichment of herbicide-responsive residues. We then targeted these herbicide-responsive regions in the rice genome using a CRISPR-directed evolution platform and identified herbicide-resistant ALS variants. Thus, our system could be used for the continuous synthetic evolution of gene functions to produce variants with improved herbicide resistance, as well as for other trait engineering applications.

Directed evolution involves generating diverse sequence variants of a gene of interest to produce a desirable trait under selective pressure. CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) systems can be programmed to target any genomic locus and perform targeted directed evolution. Here, we discuss the opportunities and challenges of this emerging platform for targeted crop improvement.

Functional genomics is at the core of studying the exact function of genes. However, homozygous knockouts of essential and pleiotropic effectors (almost 10% of the genome) are not always possible, thus, functions of these genes remain obscured. The tissue-specific genome editing tool (CRISPR-TSKO) recently described by Decaestecker et al., can characterize these indispensable genes and has wide applications in plants.

Precise genome editing by systems such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) requires high-efficiency homology-directed repair (HDR). Different technologies have been developed to improve HDR but with limited success. Here, we generated a fusion between the Cas9 endonuclease and the Agrobacterium VirD2 relaxase (Cas9-VirD2). This chimeric protein combines the functions of Cas9, which produces targeted and specific DNA double-strand breaks (DSBs), and the VirD2 relaxase, which brings the repair template in close proximity to the DSBs, to facilitate HDR. We successfully employed our Cas9-VirD2 system for precise ACETOLACTATE SYNTHASE (OsALS) allele modification to generate herbicide-resistant rice (Oryza sativa) plants, CAROTENOID CLEAVAGE DIOXYGENASE-7 (OsCCD7) to engineer plant architecture, and generate in-frame fusions with the HA epitope at HISTONE DEACETYLASE (OsHDT) locus. The Cas9-VirD2 system expands our ability to improve agriculturally important traits in crops and opens new possibilities for precision genome engineering across diverse eukaryotic species., Ali, Shami, Sedeek et al. generate a fusion between Cas9 and the VirD2 relaxase (Cas9-VirD2), which combines the functions of both proteins in producing targeted and specific double strand breaks and promoting homology-directed repair. They show the utility of their method by producing herbicide resistant rice.

Retrons are a class of retroelements that produce multicopy single-stranded DNA (ssDNA) and participate in anti-phage defenses in bacteria. Retrons have been harnessed for the overproduction of ssDNA, genome engineering and directed evolution in bacteria, yeast and mammalian cells. Retron-mediated ssDNA production in plants could unlock their potential applications in plant biotechnology. For example, ssDNA can be used as a template for homology-directed repair (HDR) in several organisms. However, current gene editing technologies rely on the physical delivery of synthetic ssDNA, which limits their applications. Here, we demonstrated retron-mediated overproduction of ssDNA in Nicotiana benthamiana. Additionally, we tested different retron architectures for improved ssDNA production and identified a new retron architecture that resulted in greater ssDNA abundance. Furthermore, co-expression of the gene encoding the ssDNA-protecting protein VirE2 from Agrobacterium tumefaciens with the retron systems resulted in a 10.7-fold increase in ssDNA production in vivo. We also demonstrated clustered regularly interspaced short palindromic repeats-retron-coupled ssDNA overproduction and targeted HDR in N. benthamiana. Overall, we present an efficient approach for in vivo ssDNA production in plants, which can be harnessed for biotechnological applications. Graphical Abstract

The SF3B complex, a multiprotein component of the U2 snRNP of the spliceosome, plays a crucial role in recognizing branch point sequence and facilitates spliceosome assembly and activation. Several chemicals that bind SF3B1 and PHF5A subunits of the SF3B complex inhibit splicing. We recently generated a splicing inhibitor-resistant SF3B1 mutant named SF3B1 GEX1A RESISTANT 4 (SGR4) using CRISPR-mediated directed evolution, whereas splicing inhibitor-resistant mutant of PHF5A (Overexpression-PHF5A GEX1A Resistance, OGR) was generated by expressing an engineered version PHF5A-Y36C. Global analysis of splicing in wild type and these two mutants revealed the role of SF3B1 and PHF5A in splicing regulation. This analysis uncovered a set of genes whose intron retention is regulated by both proteins. Further analysis of these retained introns revealed that they are shorter, have a higher GC content, and contain shorter and weaker polypyrimidine tracts. Furthermore, splicing inhibition increased seedlings sensitivity to salt stress, consistent with emerging roles of splicing regulation in stress responses. In summary, we uncovered the functions of two members of the plant branch point recognition complex. The novel strategies described here should be broadly applicable in elucidating functions of splicing regulators, especially in studying the functions of redundant paralogs in plants., Butt et al. used CRISPR-mediated directed evolution to generate rice mutants for the spliceosome components SF3B1 and PHF5A. They demonstrate that these mutants have different levels of sensitivity to salt treatments and suggest that the strategies they employed can be used in the future to study functions of redundant paralogs in plants.

Robust, sensitive, and specific diagnostic platforms for early pathogen detection are essential for the identification of infected patients and management of current and future pandemics. CRISPR-Cas systems have been repurposed for SARS-CoV-2 detection in two-pot assays. Two-pot assays require extra steps and are prone to cross-contamination; however, the temperature range of current Cas enzymes limits the development of one-pot assays Here, we characterized TccCas13a, a thermophilic Cas13a enzyme with cis and trans activities from 37–70°C, and HheCas13a, which had a limited range and lower activity. We harnessed TccCas13a in a one-pot SARS-CoV-2 assay with two layers of amplification and TccCas13a-mediated collateral degradation of a single-stranded RNA reporter. This assay showed 95% sensitivity and 100% specificity compared with RT-qPCR on clinical samples. We also developed a mobile phone application to facilitate data reading, collection, and sharing. Our OPTIMA-dx detection module exhibits key features for point-of-care SARS-CoV-2 screening and pathogen detection in general.

Efficient delivery of DNA, RNA, and genome engineering machinery to plant cells will enable efforts to genetically modify plants for global food security, sustainable energy production, synthetic biology applications, and climate change resilience. For the delivery of functional genetic units into plant cells, charged nanoparticles, particularly carbon nanotubes (CNT), have attracted considerable interest. Although some success has been achieved using CNT-based approaches, the efficiency, batch reproducibility, and the limits of their applicability remain to be assessed. Here, we provide a mechanistic understanding of plasmid DNA-loaded CNTs based transfection of plant cells, and factors affecting the expression of the transformed plasmid. We show that transfection is inherently limited by the presence of the cell wall, Coulomb interactions between DNA and polymer coated CNT, and DNA size, whereas expression of the transformed plasmid is limited by relative gene-to-plasmid size and the intracellular accessibility of DNA. We further show that the formation of partially condensed DNA on the CNT surface is a prerequisite for successful transfection and expression. Furthermore, DNA does not detach completely from the CNT, so the accessibility of the transcription machinery to DNA is the key for transformation efficiency. This irreversible DNA plasmid binding and partial condensation limit the length of DNA that can be expressed, thus negatively affecting efficiency and reproducibility. Understanding the underlying mechanisms and limitations of CNT-mediated delivery of DNA through the plant cell wall is of considerable importance in guiding efforts to design nanomaterials for efficient transformation, trait engineering, and synthetic biology applications.

Genetic variation accelerates adaptation and resilience and enables the survival of species in their changing environment. Increasing the genetic diversity of crop species is essential to improve their yield and enhance food security. Synthetic directed evolution (SDE) employs localized sequence diversification (LSD) of gene sequence and selection pressure to evolve gene variants with better fitness, improved properties and desired phenotypes. Recently, CRISPR–Cas-dependent and -independent technologies have been applied for LSD to mediate synthetic evolution in diverse species, including plants. SDE holds excellent promise to discover, accelerate and expand the range of traits of the value in crop species. Here, we highlight the efficient SDE approaches for the LSD of plant genes, selection strategies and critical traits for targeted improvement. We discuss the potential of emerging technologies, including CRISPR–Cas base editing, retron editing, EvolvR and prime editing, to establish efficient SDE in plants. Moreover, we cover CRISPR–Cas-independent technologies, including T7 polymerase editor for continuous evolution. We highlight the key challenges and potential solutions of applying SDE technologies to improve the plant traits of the value., Graphical abstract

Rapid, point-of-care (POC) diagnostics are essential to mitigate the impacts of current (and future) epidemics; however, current methods for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) require complicated laboratory tests that are generally conducted off-site and require substantial time. CRISPR-Cas systems have been harnessed to develop sensitive and specific platforms for nucleic acid detection. These detection platforms take advantage of CRISPR enzymes' RNA-guided specificity for RNA and DNA targets and collateral trans activities on single-stranded RNA and DNA reporters. Microbial genomes possess an extensive range of CRISPR enzymes with different specificities and levels of collateral activity; identifying new enzymes may improve CRISPR-based diagnostics. Here, we identified a new Cas13 variant, which we named as miniature Cas13 (mCas13), and characterized its catalytic activity. We then employed this system to design, build, and test a SARS-CoV-2 detection module coupling reverse transcription loop-mediated isothermal amplification (RT-LAMP) with the mCas13 system to detect SARS-CoV-2 in synthetic and clinical samples. Our system exhibits sensitivity and specificity comparable to other CRISPR systems. This work expands the repertoire and application of Cas13 enzymes in diagnostics and for potential in vivo applications, including RNA knockdown and editing. Importantly, our system can be potentially adapted and used in large-scale testing for diverse pathogens, including RNA and DNA viruses, and bacteria.

Facing the challenges of the world's food sources posed by a growing global population and a warming climate will require improvements in plant breeding and technology. Enhancing crop resiliency and yield via genome engineering will undoubtedly be a key part of the solution. The advent of new tools, such as CRIPSR/Cas, has ushered in significant advances in plant genome engineering. However, several serious challenges remain in achieving this goal. Among them are efficient transformation and plant regeneration for most crop species, low frequency of some editing applications, and high attrition rates. On March 8 and 9, 2021, experts in plant genome engineering and breeding from academia and industry met virtually for the Keystone eSymposium "Plant Genome Engineering: From Lab to Field" to discuss advances in genome editing tools, plant transformation, plant breeding, and crop trait development, all vital for transferring the benefits of novel technologies to the field.

In animals, distant H3K27me3-marked Polycomb targets can establish physical interactions forming repressive chromatin hubs. In plants, growing evidence suggests that H3K27me3 acts directly or indirectly to regulate chromatin interactions, although how this histone modification modulates 3D chromatin architecture remains elusive. To decipher the impact of the dynamic deposition of H3K27me3 on the Arabidopsis thaliana nuclear interactome, we combined genetics, transcriptomics, and several 3D epigenomic approaches. By analyzing mutants defective for histone H3K27 methylation or demethylation, we uncovered the crucial role of this chromatin mark in short- and previously unnoticed long-range chromatin loop formation. We found that a reduction in H3K27me3 levels led to a decrease in the interactions within Polycomb-associated repressive domains. Regions with lower H3K27me3 levels in the H3K27 methyltransferase clf mutant established new interactions with regions marked with H3K9ac, a histone modification associated with active transcription, indicating that a reduction in H3K27me3 levels induces a global reconfiguration of chromatin architecture. Altogether, our results reveal that the 3D genome organization is tightly linked to reversible histone modifications that govern chromatin interactions. Consequently, nuclear organization dynamics shapes the transcriptional reprogramming during plant development and places H3K27me3 as a key feature in the coregulation of distant genes.

We would like to thank members of the genome engineering and synthetic biology laboratory at KAUST for their critical discussion and technical help in this work.

Serine/arginine-rich (SR) proteins are conserved RNA-binding proteins that play major roles in RNA metabolism. They function as molecular adaptors, facilitate spliceosome assembly and modulate constitutive and alternative splicing of pre-mRNAs. Pre-mRNAs encoding SR proteins and many other proteins involved in stress responses are extensively alternatively spliced in response to diverse stresses. Hence, it is proposed that stress-induced changes in splice isoforms contribute to the adaptation of plants to stress responses. However, functions of most SR genes and their splice isoforms in stress responses are not known. Lack of easy and robust tools hindered the progress in this area. Emerging technologies such as CRISPR/Cas9 will facilitate studies of SR function by enabling the generation of single and multiple knock-out mutants of SR subfamily members. Moreover, CRISPR/Cas13 allows targeted manipulation of splice isoforms from SR and other genes in a constitutive or tissue-specific manner to evaluate functions of individual splice variants. Identification of the in vivo targets of SR proteins and their splice variants using the recently developed TRIBE (Targets of RNA-binding proteins Identified By Editing) and other methods will help unravel their mode of action and splicing regulatory elements under various conditions. These new approaches are expected to provide significant new insights into the roles of SRs and splice isoforms in plants adaptation to diverse stresses.

Heat stress threatens agriculture worldwide. Plants acquire heat stress tolerance through priming, which establishes stress memory during mild or severe transient heat stress. Such induced thermotolerance restructures metabolic networks and helps maintain metabolic homeostasis under heat stress. Here, we used an electrospray ionization mass spectrometry-based platform to explore the composition and dynamics of the metabolome of Arabidopsis thaliana under heat stress and identify metabolites involved in thermopriming. Primed plants performed better than non-primed plants under severe heat stress due to altered energy pathways and increased production of branched-chain amino acids, raffinose family oligosaccharides (RFOs), lipolysis products, and tocopherols. These metabolites serve as osmolytes, antioxidants and growth precursors to help plants recover from heat stress, while lipid metabolites help protect membranes against heat stress. The carbohydrate (e.g., sucrose and RFOs) and lipid superpathway metabolites showed the most significant increases. Under heat stress, there appears to be crosstalk between carbohydrate metabolism (i.e., the thermomemory metabolites stachyose, galactinol, and raffinose) and tyrosine metabolism towards the production of the thermomemory metabolite salidroside, a phenylethanoid glycoside. Crosstalk occurs between two glycerophospholipid pathways (the biosynthetic pathways of the thermomemory metabolite S-adenosyl-L-homocysteine and the terpenoid backbone) and the δ-tocopherol (chloroplast lipid) pathway, which favors the production of glycine betaine and other essential tocopherols, respectively, compounds which are essential for abiotic stress tolerance in plants. Therefore, metabolomic analysis can provide comprehensive insights into the metabolites involved in stress responses, which could facilitate plant breeding to maximize crop yields under adverse conditions.

Molecular engineering of plant immunity to confer resistance against plant viruses holds great promise for mitigating crop losses and improving plant productivity and yields, thereby enhancing food security. Several approaches have been employed to boost immunity in plants by interfering with the transmission or lifecycles of viruses. In this review, we discuss the successful application of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) (CRISPR/Cas) systems to engineer plant immunity, increase plant resistance to viruses, and develop viral diagnostic tools. Furthermore, we examine the use of plant viruses as delivery systems to engineer virus resistance in plants and provide insight into the limitations of current CRISPR/Cas approaches and the potential of newly discovered CRISPR/Cas systems to engineer better immunity and develop better diagnostics tools for plant viruses. Finally, we outline potential solutions to key challenges in the field to enable the practical use of these systems for crop protection and viral diagnostics., CRISPR-Cas systems unlock the potential of understanding the molecular basis of plant virus interactions, engineering immunity against plant viruses, and developing sensitive and specific diagnostics.

Abscisic acid (ABA) is an important phytohormone mediating osmotic stress responses. SUCROSE NONFERMENTING 1 (SNF1)-RELATED PROTEIN KINASE 2.6 (SnRK2.6, also named OPEN STOMATA1 and SNF1-RELATED KINASE 2E) is central in the ABA signaling pathway; therefore, manipulating its activity may be useful to confer stress tolerance in plants. Pladienolide B (PB) is an mRNA splicing inhibitor and enhances ABA responses. Here, we analyzed the effect of PB on Arabidopsis SnRK2.6. PB enhanced the activity of recombinant SnRK2.6 in vitro through direct physical interaction as predicted by molecular docking simulations followed by mutation experiments and isothermal titration calorimetry. Structural modeling predicted probable interaction sites between PB and SnRK2.6, and experiments with mutated SnRK2.6 revealed that Leu-46 was the most essential amino acid residue for SnRK2.6 activation by PB. This study demonstrates the feasibility of SnRK2.6 chemical manipulation and paves the way for the modification of plant osmotic stress responses.

To meet increasing global food demand, breeders and scientists aim to improve the yield and quality of major food crops. Plant diseases threaten food security and are expected to increase because of climate change. CRISPR genome-editing technology opens new opportunities to engineer disease resistance traits. With precise genome engineering and transgene-free applications, CRISPR is expected to resolve the major challenges to crop improvement. Here, we discuss the latest developments in CRISPR technologies for engineering resistance to viruses, bacteria, fungi, and pests. We conclude by highlighting current concerns and gaps in technology, as well as outstanding questions for future research. ispartof: GENOME BIOLOGY vol:21 issue:1 ispartof: location:England status: published

Highlights • RT-LAMP coupled with CRISPR-Cas12 provides a sensitive and specific virus detection platform. • iSCAN sensitivity and specificity are comparable with RT-qPCR. • iSCAN is a 1 h detection module that can help in testing in low resource areas. • iSCAN can be developed as a one-pot assay. • iSCAN reagents can be produced locally and deployed for SARS-CoV2 detection., The COVID-19 pandemic caused by SARS-CoV-2 affects all aspects of human life. Detection platforms that are efficient, rapid, accurate, specific, sensitive, and user friendly are urgently needed to manage and control the spread of SARS-CoV-2. RT-qPCR based methods are the gold standard for SARS-CoV-2 detection. However, these methods require trained personnel, sophisticated infrastructure, and a long turnaround time, thereby limiting their usefulness. Reverse transcription-loop-mediated isothermal amplification (RT-LAMP), a one-step nucleic acid amplification method conducted at a single temperature, has been used for colorimetric virus detection. CRISPR-Cas12 and CRISPR-Cas13 systems, which possess collateral activity against ssDNA and RNA, respectively, have also been harnessed for virus detection. Here, we built an efficient, rapid, specific, sensitive, user-friendly SARS-CoV-2 detection module that combines the robust virus amplification of RT-LAMP with the specific detection ability of SARS-CoV-2 by CRISPR-Cas12. Furthermore, we combined the RT-LAMP-CRISPR-Cas12 module with lateral flow cells to enable highly efficient point-of-care SARS-CoV-2 detection. Our iSCAN SARS-CoV-2 detection module, which exhibits the critical features of a robust molecular diagnostic device, should facilitate the effective management and control of COVID-19.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a powerful tool for genome engineering in plants. The RNAguided Cas9 endonuclease is usually delivered into plant cells as a DNA construct encoding Cas9 and the single guide RNA (sgRNA). However, constitutive expression of nucleases may cause off target mutations. In addition, DNA constructs can integrate into the host genome, causing mutations and complicating regulatory approval. Instead of DNA, here we deliver Cas9 through the Agrobacterium T4SS, accomplished by fusion of the VirF T4SS translocation peptide to Cas9 (NCas9F). Co-cultivation of Agrobacteria expressing NCas9F with yeast (Saccharomyces cerevisiae) harboring a sgRNA targeting CAN1 showed that NCas9F was translocated via T4SS and induced targeted mutations in the yeast genome. Infiltration of Nicotiana benthamiana leaves with Agrobacteria expressing NCas9F and sgRNA-PHYTOENE DESATURASE (PDS) resulted in targeted modifications at the PDS locus, albeit at a very low rate. In order to increase the mutation frequency NCas9F protein was co-transported with a TDNA encoding sgRNA-PDS1. Next generation sequencing confirmed that this resulted in targeted mutations at the PDS locus with a similar distribution but at a 5-fold lower frequency as the mutations obtained with a T-DNA encoding both Cas9 and sgRNA-PDS1. Similarly, infection with Tobacco rattle virus (TRV) encoding sgRNA-PDS2 combined with NCas9F protein translocation resulted in an equally high frequency of PDS mutations in N. benthamiana compared to T-DNA encoded sgRNA-PDS1 combined with NCas9F protein translocation. Our results revealed that translocation of NCas9F protein via the Agrobacterium T4SS can be used for targeted mutagenesis in host cells instead of the permanent and constitutive expression of Cas9 from a T-DNA.

The COVID-19 pandemic caused by SARS-CoV-2 affects all aspects of human life. Detection platforms that are efficient, rapid, accurate, specific, sensitive, and user friendly are urgently needed to manage and control the spread of SARS-CoV-2. RT-qPCR based methods are the gold standard for SARS-CoV-2 detection. However, these methods require trained personnel, sophisticated infrastructure, and a long turnaround time, thereby limiting their usefulness. Reverse transcription-loop-mediated isothermal amplification (RT-LAMP), a one-step nucleic acid amplification method conducted at a single temperature, has been used for colorimetric virus detection. CRISPR-Cas12 and CRISPR-Cas13 systems, which possess collateral activity against ssDNA and RNA, respectively, have also been harnessed for virus detection. Here, we built an efficient, rapid, specific, sensitive, user-friendly SARS-CoV-2 detection module that combines the robust virus amplification of RT-LAMP with the specific detection ability of SARS-CoV-2 by CRISPR-Cas12. Furthermore, we combined the RT-LAMP-CRISPR-Cas12 module with lateral flow cells to enable highly efficient point-of-care SARS-CoV-2 detection. Our iSCAN SARS-CoV-2 detection module, which exhibits the critical features of a robust molecular diagnostic device, should facilitate the effective management and control of COVID-19.

Rice (Oryza sativa) is an important staple food crop worldwide; to meet the growing nutritional requirements of the increasing population in the face of climate change, qualitative and quantitative traits of rice need to be improved. Stress-tolerant crop varieties must be developed with higher yields and increased adaptability to the changing climate. Genome editing and speed breeding have improved the accuracy and pace of rice breeding. New breeding technologies including genome editing have been established in rice, expanding the potential for crop improvement. Recently, other genome editing techniques such as CRISPR-directed evolution, CRISPR-Cpf1, and base editors have also been used for efficient genome editing in rice. Since rice is an excellent model system for functional studies due to its small genome and close syntenic relationships with other cereal crops, new genome-editing technologies continue to be developed for use in rice. In this review, we focus on genome-editing tools for rice improvement to address current challenges and provide examples of genome editing in rice. We also shed light on expanding the scope of genome editing and systems for delivering homology-directed repair templates. Finally, we discuss safety concerns and methods for obtaining transgene-free crops.

For infectious diseases, rapid and accurate identification of the pathogen is critical for effective management and treatment, but diagnosis remains challenging, particularly in resource-limited areas. Methods that accurately detect pathogen nucleic acids can provide robust, accurate, rapid, and ultrasensitive technologies for point-of-care diagnosis of pathogens, and thus yield information that is invaluable for disease management and treatment. Several technologies, mostly PCR-based, have been employed for pathogen detection; however, these require expensive reagents and equipment, and skilled personnel. CRISPR/Cas systems have been used for genome editing, based on their ability to accurately recognize and cleave specific DNA and RNA sequences. Moreover, following recognition of the target sequence, certain CRISPR/Cas systems including orthologues of Cas13, Cas12a, and Cas14 exhibit collateral nonspecific catalytic activities that can be employed for nucleic acid detection, for example by degradation of a labeled nucleic acid to produce a fluorescent signal. CRISPR/Cas systems are amenable to multiplexing, thereby enabling a single diagnostic test to identify multiple targets down to attomolar (10-18 mol/L) concentrations of target molecules. Developing devices that couple CRISPR/Cas with lateral flow systems may allow inexpensive, accurate, highly sensitive, in-field deployable diagnostics. These sensors have myriad applications, from human health to agriculture. In this review, we discuss the recent advances in the field of CRISPR-based biosensing technologies and highlight insights of their potential use in a myriad of applications.

Additional file 5: Review history.

Additional file 1. Supplementary figures.

In prokaryotes, CRISPR/Cas adaptive immunity systems target and destroy nucleic acids derived from invading bacteriophages and other foreign genetic elements. In eukaryotes, the native function of these systems has been exploited to combat viruses in mammals and plants. Rewired CRISPR/Cas9 and CRISPR/Cas13 systems have been used to confer resistance against DNA and RNA viruses, respectively. Here, we discuss recent approaches employing CRISPR/Cas systems to combat viruses in eukaryotes, highlight key challenges, and provide future perspectives. Moreover, we discuss the application of CRISPR/Cas systems in genome-wide screens to identify key host factors for virus infection to enhance our understanding of basic virus biology and to identify and study virus-host interactions.

Background CRISPR-Cas systems endow bacterial and archaeal species with adaptive immunity mechanisms to fend off invading phages and foreign genetic elements. CRISPR-Cas9 has been harnessed to confer virus interference against DNA viruses in eukaryotes, including plants. In addition, CRISPR-Cas13 systems have been used to target RNA viruses and the transcriptome in mammalian and plant cells. Recently, CRISPR-Cas13a has been shown to confer modest interference against RNA viruses. Here, we characterized a set of different Cas13 variants to identify those with the most efficient, robust, and specific interference activities against RNA viruses in planta using Nicotiana benthamiana. Results Our data show that LwaCas13a, PspCas13b, and CasRx variants mediate high interference activities against RNA viruses in transient assays. Moreover, CasRx mediated robust interference in both transient and stable overexpression assays when compared to the other variants tested. CasRx targets either one virus alone or two RNA viruses simultaneously, with robust interference efficiencies. In addition, CasRx exhibits strong specificity against the target virus and does not exhibit collateral activity in planta. Conclusions Our data establish CasRx as the most robust Cas13 variant for RNA virus interference applications in planta and demonstrate its suitability for studying key questions relating to virus biology.

Almost all biological processes involve RNA, making it crucial to develop tools for manipulation of the transcriptome. The bacterial CRISPR/Cas13 system was recently rewired to facilitate RNA manipulation in eukaryotes, including plants. We discuss here the opportunities and limitations of using CRISPR/Cas13 in plants for various types of RNA manipulation.

Thermopriming induces genome-wide differential gene expression and alternative splicing patterns, and establishes a ‘splicing memory’ that helps plants to survive subsequent and otherwise lethal heat stress., Abiotic and biotic stresses limit crop productivity. Exposure to a non-lethal stress, referred to as priming, can allow plants to survive subsequent and otherwise lethal conditions; the priming effect persists even after a prolonged stress-free period. However, the molecular mechanisms underlying priming are not fully understood. Here, we investigated the molecular basis of heat-shock memory and the role of priming in Arabidopsis thaliana. Comprehensive analysis of transcriptome-wide changes in gene expression and alternative splicing in primed and non-primed plants revealed that alternative splicing functions as a novel component of heat-shock memory. We show that priming of plants with a non-lethal heat stress results in de-repression of splicing after a second exposure to heat stress. By contrast, non-primed plants showed significant repression of splicing. These observations link ‘splicing memory’ to the ability of plants to survive subsequent and otherwise lethal heat stress. This newly discovered priming-induced splicing memory may represent a general feature of heat-stress responses in plants and other organisms as many of the key components are conserved among eukaryotes. Furthermore, this finding could facilitate the development of novel approaches to improve plant survival under extreme heat stress.

The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system has enabled efficient genome engineering in diverse plant species. However, delivery of genome engineering reagents, such as the single guide RNA (sgRNA), into plant cells remains challenging. Here, we report the engineering of Tobacco rattle virus (TRV) and Pea early browning virus (PEBV) to deliver one or multiple sgRNAs into Nicotiana benthamiana and Arabidopsis thaliana (Col-0) plants that overexpress a nuclear localization signal containing Cas9. Our data showed that TRV and PEBV can deliver sgRNAs into inoculated and systemic leaves, and this resulted in mutagenesis of the targeted genomic loci. Moreover, in N. benthamiana, PEBV-based sgRNA delivery resulted in more targeted mutations than TRV-based delivery. Our data indicate that TRV and PEBV can facilitate plant genome engineering and can be used to produce targeted mutations for functional analysis and other biotechnological applications across diverse plant species. Key message: Delivery of genome engineering reagents into plant cells is challenging and inefficient and this limit the applications of this technology in many plant species. RNA viruses such as TRV and PEBV provide an efficient tool to systemically deliver sgRNAs for targeted genome modification.

Clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated proteins (CRISPR-Cas), a groundbreaking genome-engineering tool, has facilitated targeted trait improvement in plants. Recently, CRISPR-CRISPR from Prevotella and Francisella 1 (Cpf1) has emerged as a new tool for efficient genome editing, including DNA-free editing in plants, with higher efficiency, specificity, and potentially wider applications than CRISPR-Cas9.

[EN] Alternative splicing (AS) of precursor RNAs enhances transcriptome plasticity and proteome diversity in response to diverse growth and stress cues. Recent work has shown that AS is pervasive across plant species, with more than 60% of intron-containing genes producing different isoforms. Mammalian cell-based assays have discovered various inhibitors of AS. Here, we show that the macrolide pladienolide B (PB) inhibits constitutive splicing and AS in plants. Also, our RNA sequencing (RNA-seq) data revealed that PB mimics abiotic stress signals including salt, drought and abscisic acid (ABA). PB activates the abiotic stress-and ABA-responsive reporters RD29A::LUC and MAPKKK18::uidA in Arabidopsis thaliana and mimics the effects of ABA on stomatal aperture. Genome-wide analysis of AS by RNA-seq revealed that PB perturbs the splicing machinery and leads to a striking increase in intron retention and a reduction in other forms of AS. Interestingly, PB treatment activates the ABA signaling pathway by inhibiting the splicing of clade A PP2C phosphatases while still maintaining to some extent the splicing of ABA-activated SnRK2 kinases. Taken together, our data establish PB as an inhibitor and modulator of splicing and a mimic of abiotic stress signals in plants. Thus, PB reveals the molecular underpinnings of the interplay between stress responses, ABA signaling and post-transcriptional regulation in plants., We wish to thank members of the Laboratory for Genome Engineering at King Abdullah University of Science and Technology for helpful discussions and comments on the manuscript. We wish to thank Moussa Benhamed for helpful discussions and suggestions and for providing key materials. We wish to thank Sean Cutler for providing Arabidopsis seeds of MAKPKKK18-uidA. This study was supported by King Abdullah University of Science and Technology. Work in PR's laboratory was funded by grant BIO2014-52537-R from MINECO. Work in PD's laboratory is funded by grant PTDC/BIA-PLA/1084/2014 from FCT. The authors declare no conflicts of interest.

CRISPR/Cas9 system confers molecular immunity in archeal and bacterial species against invading foreign nucleic acids. CRISPR/Cas9 system is used for genome engineering applications across diverse eukaryotic species. In this study, we demonstrate the utility of the CRISPR/Cas9 genome engineering system for drug target validation in human cells. Pladienolide B is a natural macrolide with antitumor activities mediated through the inhibition of pre-mRNA splicing. To validate the spliceosomal target of Pladienolide B, we employed the CRSIPR/Cas9 system to introduce targeted mutations in the subunits of the SF3B complex in the HEK293T cells. Our data reveal that targeted mutagenesis of the SF3b1 subunit exhibited higher levels of resistance to Pladienolide B. Therefore, our data validate the spliceosomal target of Pladienolide B and provide a proof of concept on using the CRISPR/Cas9 system for drug target identification and validation., Highlights • CRISPR/Cas9 system serves as an excellent tool for drug target validation. • Pladienolide B an antitumor macrolide known to bind to the SF3b complex and inhibit pre-mRNA splicing. • Use of CRISPR/Cas9 system to confirm and validate the Pladienolide B spliceosomal target. • Our data confirm that the SF3b1 is a specific target for Pladienolide B.